Lid for pcr vessel comprising probes permitting pcr amplification and detection of the pcr product by hybridisation without opening the pcr vessel

ABSTRACT

The present invention proposes a simple and effective lid being part of a device having multiwells for performing simultaneous amplifications by PCR and detections of multiple target molecules on unlabeled capture molecules immobilized on the lid.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention proposes a simple and effective lid being part ofa device having multiwells for performing simultaneous amplifications byPCR and detections of multiple target molecules on unlabeled capturemolecules immobilized on the lid.

The invention also proposes such a lid carrying capture molecules toperform the detection and quantification of multiple nucleotidemolecules in a real-time PCR amplification.

2. Description of the Related Art

Detection and quantification of an organism, or a part of it such as agene, is best performed using the molecular amplification of thenucleotide sequence followed by its detection. Amplification of a givensequence is performed by several methods such as the polymerase chainreaction (PCR) (U.S. Pat. Nos. 4,683,195 and 4,683,202), ligase chainreaction (LCR) (Wu and Wallace, Genomics, Vol. 4, p. 560, 1989) or theCycling Probe Reaction (CPR) (U.S. Pat. No. 5,011,769) which are themost common.

PCR is the most commonly used method of amplification. PCR uses twooligonucleotide primers, an agent for polymerization, a target nucleicacid template. The successive cycles of denaturation of nucleic acid,annealing and extension of the primers produce large number of copies ofa particular nucleic acid segment. Segments of genomic DNA can beamplified up to 10 million fold with very high specificity and fidelitywhen using optimized conditions.

The PCR are usually performed in a single tubes and in wells being partof a 96-wells or 384 well plate format and are thereafter analyzed forthe presence of the amplified sequence known as amplicons.

Methods for detecting PCR products are described in U.S. Pat. No.4,683,195. These methods require an oligonucleotide probe capable ofhybridizing with the amplified target nucleic acid. These methodsrequire separate steps of amplification, capture, and detection andgenerally require several hours for completion.

Due to the large amplification of the PCR process, small levels of DNAcarryover from samples with high DNA content, or from previousamplifications, can produce non specific amplicons even in the absenceof purposefully added template DNA. Because the possibility ofintroducing contaminating DNA to a sample increases with the increasednumber of handling steps required for sample preparation, processing,and analysis is increased, it is preferable to minimize sample handlingfor their detection and quantification, particularly after theamplification reaction is complete.

Methods and devices for simultaneous amplification and detection oftarget nucleic acids have been described aimed at minimizing theproblems of sample contamination.

One particular way to detect the presence of a given target nucleic acidsequence, and thus of a particular organism, is to monitor theappearance of amplicons during the PCR cycles. The method is called realtime PCR. The method gives the possibility of quantifying the amplifiedsequence as the cycles progress, and to calculate the amount of thesequence in the original sample. The method uses a homogeneous format,and the PCR and the detection are performed within one tube. Performingboth amplification and detection in a closed chamber lowers thecontamination risk caused by opening the tubes in conventional post-PCRdetection methods.

One way to assay for the presence of the amplicons is to take advantageof certain intercalating dyes, the fluorescence of which increases, orchanges its parameters, when the dye is intercalated into doublestranded DNA. One to the most commonly used dye is SYBR green. Themethod assays for the amount of double stranded DNA, mainly theamplicons present in the solution. U.S. Pat. No. 4,683,195 and U.S. Pat.No. 6,171,785 also use the introduction of detectable DNA binding agents(such as ethidium bromide) into the amplification reaction, which agentsproduce a detectable signal in the PCR solution that is enhanced uponbinding double-stranded DNA. An increase in fluorescence of the PCRmixture indicates that amplification has occurred. In order to be usefulthe amplification has to be very specific since non specificamplifications will also lead to a signal.

U.S. Pat. No. 6,814,934 also proposed an instrument for the detection inreal-time of the fluorescent increase occurring in the solution mixture,resulting from the formation of double stranded amplicons during the PCRcycles.

The best detection method for PCR product available to date is based onthe use of probes specific of the amplicons, of which the fluorescencevaries or is released when the amplicons are formed or are present inthe solution. Early methods for detecting PCR products have beendescribed in U.S. Pat. No. 4,683,195. These methods require anoligonucleotide probe capable of hybridizing with the amplified targetnucleic acid. These methods require separate steps of amplification,capture, and detection and generally require several hours to becompleted.

New methods have been developed, including the molecular beacon probes,the double-dye oligonucleotide probes, the Amplifluor or the Scorpionsprimers and TaqMan probes. Molecular beacons are structurally similar tothe tradition single strand hybridization probe except that the ends ofthe beacon contain equal length self-complementary segments, which, inthe free state, will bond to each other forming a loop terminated by ablunt end stem. The ends of the stem have a fluorescer attached to oneside and a quencher attached to the other side, so that it is selfquenching in the unbound state. When the beacon attaches to a targetamplicon, the fluorophor and quencher become separated in space allowingthe sample to fluoresce. The TaqMan probes are among the most popularspecific methods of detection in real time and are commerciallyavailable. The TaqMan probe contains both a fluorescer and a quencher inclose proximity to one another which inhibits fluorescence in the probehybridized to the target amplicon or in excess probes in solution. Thefluorochrome is released from the vicinity of the quencher by thedigestion of the probes during the copying of the amplicon strand by theTaq polymerase having a 5′-3′ exonuclease activity. They provide adetectable molecule that accumulates in the solution during thesuccessive cycles.

The use of linear (TaqMan probe) or hairpin (molecular beacon) probeshaving a quencher and a fluorescer molecule has some importantdrawbacks. First, the probe is a complicated molecule to synthesize andis expensive. Second, a different fluorescer is necessary for eachamplicon to be quantified. This feature limits the number of ampliconspossibly detected in the same assay. Third, the distance between thefluorescer and the quencher is crucial to a have an effective quenchingof the fluorescer for the free probe. The presence of a secondarystructure in the probe may affect the distance between the fluorescerand the quencher and, as a consequence, the free probe is not properlyquenched.

U.S. Pat. No. 5,716,784 provides an alternative method based on the useof two complementary probes, a first, analytical probe being labeled atits 5′ terminus with an energy transfer donor fluorophore, and a second,detection probe being labeled at its 3′ terminus with an energy transferacceptor fluorophore. Quantitative detection of oligonucleotideanalytical probe hybridized in solution to the oligonucleotide detectionprobe provides a measure of the amount of oligonucleotide analyticalprobe used up in the amplification of the target nucleic acid sequenceand thus provides a measure of amount of target nucleic acid sequenceamplified in the PCR replication procedure. The quantitative detectionof the analytical probe involves spectrophotometrical energy transferdetection in solution.

U.S. Pat. No. 5,928,907 describes an apparatus for monitoring theformation of a nucleic acid amplification reaction product in real timethat uses an optic fiber focused in the volume of the sample. Thefluorescence is usually detected in the solution trough the tip or thebottom of the tubes or the wells.

Although these methods are capable of monitoring in real time thequantification of nucleic acids in a homogeneous PCR hybridizationsystem, they are limited to the quantification of one target nucleicacid per fluorescent dye. Multiplexing is not easy to implement due tothe fact that the detector has to be able to detect as many fluorescentdyes as there are targets or standards in the solution. This requiresthe use of non overlapping fluorescent dyes for measuring the increasein signal related to the amplification of several individual targetnucleic acid sequences in the same apparatus. In most of theapplications, one fluorescent probe is used, and sometimes 2. The use ofa larger number of probes would lead to a dramatic increase in thecomplexity and cost of the detection system since each probe requiresspecific excitation and the emission wavelengths.

WO 04/101733 discloses a wash-free PCR amplification tube for directgene detection. A molecular beacon is immobilized inside the reactiontube that is designed for the PCR purpose. The PCR tube also comprises atransparent window at the section where the molecular beacon is fixedinside the PCR tube. The immobilized probe comprises a fluorescer and aquencher. The quencher is positioned at the free end of the probe.During the PCR, the quencher is released from the vicinity of thefluorescer by the digestion of the immobilized probes during the copyingof the amplicon strand by the Taq polymerase having a 5′-3′ exonucleaseactivity. They provide a detectable molecule that accumulates on thesupport during the successive cycles.

This method is very close to the real-time PCR performed in solution. Inboth cases, the probe parties involved in the amplification step.

The use of a molecular beacon as capture molecule (immobilized on asupport) presents further drawbacks as compared to the molecular beaconused in solution. As the PCR is performed on a probe that is in theproximity of a solid support, it is much less efficient and moredifficult to implement than a PCR performed in solution. The situationis even more dramatic when multiple molecular beacons are used on thesame support, such as arranged in a micro-array, to quantify differenttargets, because the basic fluorescent background (without target) isdifferent from probe to probe. As a result the quantifications of thedifferent targets are difficult to calibrate.

The present invention aims to provide a device for a method that has theadvantages of the real time PCR methods described here above usingspecific probes for the detection, but that overcomes the limitations ofsingle (or very few) detection per assay and/or the complexity of thedesign of labeled probes having both specificity and physico-chemicalconstraints such as quenching or FRET.

BRIEF SUMMARY OF THE INVENTION

The present invention aims to overcome most of the above mentionedlimitations by proposing a simple and effective lid being part of adevice having multiwells for performing simultaneous amplifications byPCR and detections of multiple target molecules on unlabeled capturemolecules immobilized on the lid.

In order to realize the above-mentioned objectives, the inventionrelates to a sealable lid (1) for a PCR multiwell plate (2) providedwith at least one defined area (3), said defined area being configuredto be mated to at least one well (4) of the multiwell plate (2), atleast one of said defined area (3) being pretreated to be provided withunlabeled capture molecule (5). The lid allows real-time PCR to beperformed both in parallel assays and in multi-detections per assay.

The sealable lid (1) is also provided with at least one defined area(3), said defined area being configured to be mated to at least fourwells (4) of the multiwell plate (2), at least one of said defined area(3) being pretreated to be provided with unlabeled capture molecule (5).

One feature of the invention is that the capture molecules are nottaking part in the PCR reaction and thus do not interfere with the PCRhappening in the solution.

BRIEF DESCRIPTION OF THE DRAWINGS

The features and advantages of the invention will be appreciated uponreference to the following drawings, in which:

FIG. 1 is a general schematic representation of a preferred sealable lid(1) according to the invention, comprising a plurality of defined areas(3), each defined area corresponding to at least one well (4) of themultiwell plate (2) on which it can be mated by a sealable material (9).

FIG. 2 a is a general schematic representation of a preferred sealablelid (1) being configured to be mated to a multiwell plate by means of asealable material (9).

FIG. 2 b shows a detailed top view of a defined area (3) carryingimmobilized capture molecules (5).

FIG. 2 c shows another embodiment for the defined area (3) being dividedin four compartments (6), each compartment carrying different capturemolecules (5, 5′, 5″, 5″′).

FIG. 2 d shows an alternative embodiment for the defined area (3)carrying capture molecules being immobilized according to a micro-array(7) in specifically localized areas (8).

FIG. 3 is a general schematic representation of a preferred sealable lid(1) according to the invention, comprising a plurality of defined areas(3), each defined area corresponding to at least four wells (4) of themultiwell plate (2) on which it can be mated by a sealable material (9).This lid is preferably used for end point measurement of PCR product.After the PCR, the multiwell plate (2) is flipped and the content offour wells comprising four different PCR products is contacted with onedefined area (3) carrying immobilized capture molecules (5).

FIG. 4. Results for the end point measurement of SNP (single nucleotidepolymorphism) in PCR products using a preferred lid of the invention asprovided in FIG. 3. Three PCR were performed in individual tubes of a96-wells plate using cy3 labeled primers. Each tube contained adifferent SNP of the CYP2C9 gene: mutation 3 of exon 7, mutation 10 andcorresponding wild type sequence of exon 5 and the corresponding primerpairs. A lid comprising a defined area having fixed upon its surface amicroarray was fixed on the top of the three PCR tubes by means of anadaptor. The microarray contained the different capture moleculesspecific of the three amplicons (2C9*3 for mutation 3 of exon 7; 2C9*1,10 for wild type sequence of exon 5 and 2C9*10 for mutation 10 of exon5). In addition, we used a control capture molecule having a substitutedbase at the location of the mutation 3 of exon 7 (2C9*1,3) and anegative hybridization control (neg hyb ctl). After the PCR, themultiwell plate was flipped and the content of three wells comprisingthree different PCR products was contacted with the defined areacarrying immobilized capture molecules. After hybridization, themultiwell plate was flipped back, centrifuged to remove the PCR solutionfrom the lid and the micro-array was read in fluorescence using aconfocal scanner. The result shows signals on the specific capturemolecules 2C9*3, 2C9*1,10 and 2C9*10 as expected.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The following is a description of certain embodiments of the invention,given by way of example only and with reference to the drawings.

Definitions.

The terms “nucleic acid”, “oligonucleotide”, “array”, “probe”, “targetnucleic acid”, “bind substantially”, “hybridising specifically to”,“background”, and “quantifying” are as described in the internationalpatent application WO97/27317, incorporated herein by reference.

The terms “nucleotide triphosphate”, “nucleotide”, and “primer sequence”are as described in the document WO00/72018, incorporated herein byreferences.

The terms “Homologous sequences” and “consensus sequence” are describedin the European patent application WO01/77372, incorporated herein byreference. The term “homology” is intended to mean the degree ofidentity of one polynucleotide sequence to another polynucleotidesequence. There may be complete homology (i.e. 100% identity) betweentwo or more polynucleotides. The degree of homology is calculated afteralignment of the sequence and may be determined by any method well knownto a person skilled in the art.

As used herein, “capture molecule” refers to a molecule, or complex orcombination of molecules, that is capable of specifically binding to onetarget molecule, or to a family of target molecules, or to one or moremember (s) of a plurality of target molecules, or portion(s) thereof.The capture molecules are preferably nucleic acids, which are eithersynthesized chemically in situ on the surface of the support orsynthesized ex situ and subsequently affixed to the support. Nucleicacid binding is achieved via base pairing between two polynucleotides,one being the immobilized capture molecule and the other one the targetto be detected. Capture molecules also comprise derivatives of thenucleic acid, such as PNA or LNA, as long as they can bind specificallythe target polynucleotide molecule.

The term “single capture molecule species” is a composition of relatedpolynucleotides for the detection of a given sequence by base pairinghybridization or by molecular recognition between polypeptides orproteins. Polynucleotides are synthesized either chemically orenzymatically, or isolated from samples, but the synthesis or isolationis not always perfect and the capture molecule is contaminated withother related molecules, like shorter polynucleotides. The essentialcharacteristic of one capture species for the invention is that theoverall species can be used for capture of a given target nucleotidemolecule.

The term “polynucleotide sequences that are complementary to one or moregenes or to the genome sequence” described herein, refers topolynucleotides that are capable of hybridizing under stringentconditions to at least part of the nucleotide sequence of said genes orgenome or copy thereof. Polynucleotides also include oligonucleotides(comprising more than 2 bases but fewer than 100 bases), which can beused under particular conditions. Such hybridizable polynucleotides willtypically exhibit at least about 75% sequence identity at the nucleotidelevel to said genes or genome, preferably about 80% or 95% sequenceidentity or preferably more than 95% nucleotide sequence identity tosaid genes or genome. They are composed of either small sequencestypically 15-30 bases long, or longer ones being between 30 and 100 oreven longer, between 100 and 800 bases long, depending on thespecificity and sensitivity requirements for the assay.

“Micro-array” means a support on which multiple capture molecules areimmobilized in order to be able to bind to the given specific targetmolecule. The micro-array is preferentially composed of capturemolecules present at specifically localized areas on the surface orwithin the support or on the substrate covering the support. Aspecifically localized area is the area of the surface that containsbound capture molecules specific for a determined target molecule. Thespecific localized area is either known from the method that was used inbuilding the micro-array, or is defined during or after the detection. Aspot is the area where specific target molecules are fixed on theircapture molecules and seen by the detector. The micro-array may alsocontain detection controls which are labeled. These controls check theperformance of the detection and circumvent the array, but they are notused to detect specific target molecules. In one particular applicationof this invention, micro-arrays of capture molecules are also providedon different supports, as long as the different supports containspecific capture molecules and may be distinguished from each other inorder to be able to quantify the specific target molecules. This can beachieved by using a mixture of beads having particular features andbeing able to be distinguished from each other in order to quantify thebound molecules. One bead or a population of beads is then considered asa spot having a capture molecule specific of one target molecule.

The “amplicon” of the invention means target nucleotide molecules beingthe result of PCR amplification of a nucleotide molecule present in abiological material.

“Intermittent contact” means to be physically in contact or notaccording to some time frame. In a particular aspect of the inventionthe PCR solution is in intermittent contact with the capture molecule,means the PCR solution is moved or displaced from the surface havingfixed the capture molecules for a given time period (non contact) andthen it is moved back to its original position (contact). Preferablymore than 95% and preferably more than 99% of the PCR solution is movedor displaced in the reaction chamber. The PCR solution is preferablydisplaced by gravity drain resulting from a change in orientation of thereaction chamber, preferably rotation, translation, or lateral movementof the reaction chamber.

DESCRIPTION OF A PREFERRED EMBODIMENT

The lid of the invention is specifically designed to perform an endpoint measurement of PCR product or a real-time measurement of the PCRafter being sealed on a multiwell plate. The lid comprises at least onedefined area that is pretreated to be provided with capture molecules orhaving capture molecules immobilized by a consumer depending on hisrequirement. Home made immobilization is performed on pretreated lid forthe fixation of the desired capture molecules. The capture molecules arespecifically designed for the detection of amplified target nucleotidemolecules.

The lid contains at least 2, preferably at least 6, more preferably atleast 24, still more preferably at least 96 defined areas. The multiwellplate comprises preferably at least 2, preferably at least 8, morepreferably at least 24, still more preferably at least 96, even morepreferably at least 384 wells. Wells are separated from each other by apitch of 4.5, or a multiple thereof, such as 9 mm. The multiwell plateis a preferred format for performing different PCR assays in parallel.Multiplexing is obtained by using different wells carrying different PCRproducts that are contacted with different defined areas of the lidcarrying one or more capture molecule(s). The number of capturemolecules per defined area can be adapted according to the consumerrequirement.

In a preferred embodiment, the defined area of the lid has fixed uponits surface at least 4 different capture molecules that are physicallyseparated in compartments. In another preferred embodiment, the definedarea of the lid has fixed upon its surface a micro-array comprising atleast 5 different capture molecules being immobilized in specificallylocalized areas of said defined area. Preferably, the micro-arraycomprises more than 10 different capture molecules, preferably more than20 capture molecules, more preferably more than 50 capture molecules.

In still another preferred embodiment, different defined areas of thesame lid have fixed the same capture molecules.

In a preferred embodiment, capture molecules are polynucleotides used todetect target nucleotide molecules that are labeled during the PCR.

Also, the capture molecules preferably have an amplicon-specific bindingsequence (capture portion), and a spacer portion bound to the chambersurface. The immobilized capture molecule is preferably a polynucleotidehaving an amplicon-specific binding sequence, and a spacer portion of atleast 20 nucleotides, preferably at least 50, more preferably more than90 nucleotides.

Advantageously, the measurement of the amplified target nucleotidemolecules is performed on the lid of the multiwell plate afterhybridization on capture molecules.

Advantageously, the lid is not removed for the detection and there is nowashing before the measurement. The assay of amplification and detectionis performed in a closed well of the multiwell plate. Washing is avoidedsince it would include liquid handling of the solution containingamplified target and may entail possible contamination of furtherassays.

In a preferred embodiment, the measurement of the bound labeledamplicons is performed in the absence of liquid. Preferably, the absenceof liquid is obtained by gravity drain, such as resulting from changingthe orientation of the reaction chamber. For example, changing theorientation of the reaction chamber by a method selected from the groupconsisting of: rotation, translation, flipping or lateral movement ofthe reaction chamber.

One defined area of the lid is exactly aligned with one well or withmore than one well depending on the application.

In a preferred embodiment, the lid is used for real-time PCRmeasurement. The measurement of the target bound to the lid is repeatedduring at least two thermal cycles, preferably during the annealingtemperature step of the cycle. Preferably the detection is performed ineach of the PCR cycles. Advantageously, and contrary to the real-timePCR performed in homogeneous phase, there is no requirement fordifferent fluorescent dyes to quantify different nucleotide molecules.One fluorescent dye is sufficient for the quantification of multipledifferent target nucleotide molecules since they are individualized onthe defined area of the lid because of their specific binding byhybridization on capture molecules being specific of each targetnucleotide sequence and being localized in distinct areas of the definedarea of the lid (compartment or spot of a micro-array). When severalnucleotide molecules are amplified in the same solution using the sameor different primers, both amplicons will be labeled with the samefluorescent dye but they will be detected and/or quantified on separatedcapture molecules having a different position on the lid without theneed of several fluorescent dyes as required in the real time PCRperformed in solution.

In another preferred embodiment, the lid is used for end point PCRmeasurement. The measurement is performed only at the end of theamplification. In this application, one defined area of the lidcorresponds to one or more wells. In a preferred embodiment, eachdefined area corresponds to at least four wells of the multiwell plate.This is particularly advantageous in case of multiplex PCR. The use ofmore than 5 primer pairs in a PCR well often decrease the efficiency ofthe amplification due to the formation of primer dimers and non-specificannealing of the primers. The PCR can be split in different wells, eachwell having a limited number of primer pairs. At the end of theamplification, the PCR products are pooled and hybridized on the samecapture molecules being present on a single defined area of the lid.This embodiment is performed at best by flipping the multiwell plate inorder to pool the content of the separated wells and contacting the mixwith one defined area of the lid for hybridization.

Advantageously, and contrary to end point measurement of PCR productsperformed in the state of the art, there is no requirement for openingthe multiwell plate for hybridization and detection, thus avoidingsubsequent manipulation and carry over contamination.

Another advantage is that primer dimers or non-specific amplifiedproducts formed during the PCR amplification will not generate a signalon the lid since no complementary capture molecules for the primers norfor unspecific products are present on the lid.

The specificity can still be increased further by the use of differentcapture molecules for the same target nucleotide molecule. Two or morecapture molecules can be designed to bind the same strand or one capturemolecule may be provided to bind the sense strand of the amplifiedproduct and another capture molecule the antisense strand.

Advantageously, the nucleotide molecules to be amplified are homologousnucleotide sequences that are quantified on micro-array during the PCRusing consensus primers as described in WO0177372. The same primers areused to amplify all the homologous sequences which are possibly presentin a sample. The amplicons are discriminated on different capturemolecules, each one targeting a different homologous sequence. So withonly one primer pair and one fluorescent dye, the assay is mademultiplex by the use of consensus primers for the PCR and multiplecapture molecules present on the micro-array for their detection.

In a preferred embodiment, the capture molecules are attached preferablyby covalent link in the defined area of the lid. In a preferredembodiment, the capture molecules are terminated by a primary aminogroup and are covalently fixed on the defined area having fixed upontheir surface aldehyde groups. Such reaction does not require anycoupling agent and results in the formation of imine covalent link. Thislink can be further stabilized into amine function by a reducing agentpreferably in the presence of a solution of NaBH₄.

Deposition of the capture molecules on the lid can be performed by acompany upon request or by the consumer himself. Deposition of thecapture molecules by the consumer is preferentially done by pipettingthe capture molecules solutions in different compartments of the maindefined area. In a preferred embodiment, the defined area is divided inat least 4 compartments. Deposition of the capture molecule in the formof a micro-array is preferentially done with physical means such asplain pin or split pin or “pin and ring” having a physical contact withthe surface, or by release of a micro-droplet of solution by methodssuch as piezo or nanodispenser.

Alternatively, in situ synthesis of capture molecules is performed onthe support using light directed chemical synthesis for the synthesis ofoligonucleotides or polynucleotides in known locations such as providedby U.S. Pat. Nos. 5,744,305 and 6,346,413.

In a particular embodiment, the lid surface is activated in order toprovide reactive groups for the fixation of capture molecules. Thepretreated lid preferentially bears aldehyde, epoxide orN-hydroxymaleimide or any chemical group on which a capture moleculewill react by chemical reaction with such as but not limited to freeamino or sulfhydryl groups. Pretreated lids have the same embodiments asdescribed here above.

According to the invention, the lid comprises or is made of a materialselected from the group consisting of glass, metal, polymer (preferablythermo-resistant having low self-fluorescence) or any other materialused in the micro-array technology (preferably activated glass bearingaldehyde or epoxide or acrylate groups), said lid optionally furthercomprising also specific coatings, markers or devices (bar codes,electronic devices, etc.) for improving the assay.

Although glass presents many advantages (like being inert and having alow self-fluorescence), other supports such as polymers, with variouschemically well-defined groups at their surface, allowing the binding ofthe nucleotide sequences are useful. In another preferred embodiment,the support bearing the capture molecules has a 3 dimensional porousstructure. Conventional glass slides have less than 60% silicon dioxideon their surface. This inherently limits the amount of chemical bondingavailable on the surface. Porous material exhibits an increased loadingcapacity of capture molecules. Typical porous supports include gel pads,fused-fiber matrix and fibrous polymer matrix. The capture molecules canbe immobilized entirely in the porous material, or on a layer of porousmaterial mounted on top of a flat surface such as glass, plastic, ormetal.

Besides glass, polymers are becoming increasingly used as support formicro-array and for the miniaturisation of the biological assays due tothe development of the microfluidic technology and the “lab on a chip”concept. In a preferred embodiment, the polymer material of the lid isselected from the group consisting of: polycarbonate (PC), polyethylene(PE), Cycloolefin copolymer (COC), cyclic olefin polymer (COP) or amixture thereof. A preferred COP product is Zeonex® because of itsexcellent optical properties, chemical resistance, thermal stability andlow fluorescence (http://www.zeonchemicals.com).

In a preferred embodiment, the defined area of the lid is configured tobe mated to at least one well of a multiwell plate by means of asealable material. The sealable material is preferably made of a softpolymer or a glue which surrounds each defined area in order to assure aperfect sealing to the corresponding well of the multiwell plate.

In another preferred embodiment, the defined area is configured to bemated to at least one well of the multiwell plate by using an adaptorcomprising a sealable material. The adaptor is preferably made of a softpolymer which is placed between the lid and the multiwell plate andassures a perfect sealing between a defined area of the lid and thecorresponding well of the multiwell plate. If necessary, pressure can beapplied to the lid to obtain a sealing with the multiwell plate.

In another preferred embodiment, the surface of a defined area of thelid bearing the capture molecule is maintained flat at temperaturehigher than 85° C., preferably higher than 95° C. The hybridizedamplicons have to be detected by a detector and preferably the differentlocalized area that contain different capture molecules and present in adefined area have to show the same signal intensity if bound with thesame amount of targets. Thus in a preferred embodiment a defined area ofthe lid has a flatness tolerance of less than 100 microns preferably ofless than 25 microns.

In a preferred embodiment, the lid is thermo-resistant and is maintainedflat at temperature higher than 85° C. In another preferred embodiment,the light transmittance of the lid at the wavelength used for thedetection is higher than 80% and even higher than 90%.

In another preferred embodiment, the signal detected trough the windowresulting from the binding of the amplicons to the immobilized capturemolecules is at least 2 times, preferably at least 5 times, morepreferably at least 10 times higher than the signal obtained in theabsence of amplicons or in conditions in which no binding can takeplace.

In the preferred embodiment, the polynucleotides being used as capturemolecule are between 10 and 1000 nucleotide long and preferably between100 and 400 nucleotides long. For specific binding of homologoussequences possibly present in the same sample, the polynucleotidecapture molecules contain a spacer portion according to the patentWO0177372. Specific binding of homologous sequences or SNP possiblypresent in the same sample, are obtained using capture molecules havinga specific part of between 10 and 30 nucleotides.

In a preferred embodiment, the polynucleotides that are used as capturemolecules are present on the micro-array localized area at a densitysuperior to 10 fmoles per cm², and preferably more than 100 fmoles percm² surface of the solid support.

The micro-array according to this invention contains between 4 and100000 spots per cm² and preferably between 20 and 1000 spots per cm².Each spot is preferably the localized area for one capture molecule.Miniaturization allows performing one assay upon a large number ofsurface spots (usually circular spots of about 0.1 to about 1 mmdiameter). A low density array, containing 20 to 400 spots is easilyobtained at low cost with pins of between 0.2 and 0.4 mm of diameter.Higher density of spots up to 1,600 spots per cm2 can be obtained byreducing the size of the spots for example between 0.1 mm and 0.2 mmdiameter. Methods for obtaining capture molecules of higher density havebeen described earlier as in U.S. Pat. No. 5,445,934. Miniaturization ofthe spot size allows for a high number of data to be obtained andanalyzed simultaneously, the possibility to perform replicates and withonly a small amount of biological sample being necessary for the assay.Miniaturization for detection on micro-arrays is preferably associatedwith microfluidic substrate for separation, extraction of nucleotidemolecules from a cell extract.

In a preferred embodiment, the localized area is comprised between about10 μm² and about 1 mm² and preferably between about 1 μm² and about 100μm².

In one preferred embodiment, the capture molecules present on the lidare complementary to at least one part of the sequence of an amplifiedtarget nucleotide sequence present in solution. The capture moleculescomprise a nucleotide sequence that is able to specifically bind theamplified target nucleotide sequence, said specific nucleotide sequence(capture portion) is also preferably separated from the surface of thesolid support by a spacer arm (spacer portion) of at least about 6.8 nmor 20 nucleotides in a double stranded form, and which has no bindingaffinity for the amplified target molecule. In a preferred embodiment,the capture molecule is a single stranded polynucleotide containing acapture portion able to specifically bind the labeled target nucleotidemolecule and a spacer portion of at least 20 nucleotides and preferablymore than 90 nucleotides. The spacer portion can be either single ordouble stranded DNA.

In a preferred embodiment the capture portion of the capture molecule iscomprised between 15 and 100 nucleotides and more preferably between 15and 35 nucleotides.

Detectable labels suitable for use in the present invention include anycomposition detectable by electromagnetic light emission. In a preferredembodiment, the target molecules are labeled with a fluorescent dye. Thefluorescent label is preferably incorporated into the target byenzymatic or chemical reaction. Typical enzyme reaction includes theincorporation of nucleotide analogues into the target. Alternatively,primers labeled at their 5′ end with a fluorescent dye are incorporatedinto the target. Fluorochromes are also incorporated into the targets bychemical reaction such as the reaction of fluorescent dye bearing aN-hydroxysuccinimide (NHS) group with amines groups of the targets.Useful fluorescent dyes in the present invention include Cyanine dyes(Cy3, Cy5, Cy7), Fluorescein, Texas red, Rhodamine, Green FluorescentProtein. Preferably, the excitation wavelength for Cyanin 3 is comprisedbetween 540 and 558 nm with a peak at 550 nm and the emission wavelengthis comprised between 562 and 580 nm with a peak at 570 nm.

Preferably, the excitation wavelength for Cyanin 5 is comprised between639 and 659 nm with a peak at 649 nm and the emission wavelength iscomprised between 665 and 685 nm with a peak at 670 nm. Preferably, theexcitation wavelength for Cyanin 7 is comprised between 733 and 753 nmwith a peak at 743 nm and the emission wavelength is comprised between757 and 777 nm with a peak at 767 nm.

Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and4,366,241. In a preferred embodiment, the fluorescent dye is Cyanin 3,Cyanin 5 or Cyanin 7.

The original nucleotide molecule is not necessary labeled in the samplebut should lead to amplified labeled target molecules during theamplification step. The amplified nucleotide molecules are able tohybridize on the capture molecules after a denaturation step.

As the amplified nucleotide molecules are double stranded, in theorythey should reassociate in solution much faster than they hybridise oncapture molecules fixed on a solid support where diffusion is low andthe specific binding sequence is short, thus reducing even more the rateof reaction. Therefore, it was unexpected to observe a significantsignal increase on the capture molecules over multiple thermal cyclesafter a short period of incubation time.

In a particular embodiment the measurement is performed on bound targetlabeled molecules present on the lid while they reassociate in a doublestranded form in the solution during annealing and/or elongation of thethermal cycle.

Advantageously, the length of the amplified target nucleotide moleculesare selected as being of a limited length preferably between 100 and2000 bases, preferably between 200 and 1500 bases, and still morepreferably between 300 and 800 bases. This preferred requirement dependson the possibility to find primers to amplify the required sequencespossibly present in the sample. Too long target may reallocate fasterand adopt secondary structures which may inhibit the fixation on thecapture molecules.

The thermal cycler is adapted to fit the support format of a 96 wellsmultiwell plate.

The alternative heating and cooling is preferably obtained using aPeltier element or pulsed air.

In a preferred embodiment, the light beam is a laser beam which isfocused on the surface of the lid defined area in order to excitedirectly the fluorescent molecules. The laser beam is preferably focusedperpendicular to the surface of the lid. The emitted light is detectedin the opposite direction of the excitation laser beam. The emittedlight is preferably detected as a confocal light and measured afteramplification by a photomultiplier. In a preferred embodiment thesurface of the lid defined area is scanned by the laser beam in order toobtain a maximum light excitation of the bound targets.

In a preferred embodiment, the signal associated with a capture moleculeon the lid defined area is quantified. The preferred method is thescanning of the array(s) with a scanner which is preferably a laserconfocal scanner for the detection of fluorescent labeled targets. Theresolution of the image is comprised between 1 and 500 μm and preferablybetween 5 and 50 μm.

The lid defined area is preferably scanned and each localized area ofthe micro-array or each compartment is subsequently measured. Preferablythe scanning of the array is performed within 1 min, more preferablywithin 30 sec and still more preferably within 10 sec. If reading isrepeated over multiple thermal cycles, the scan of each localized areapreferably is measured at the same precise moment of a temperature step.

A subsequent measurement of different defined areas of the lid can beadvantageously used to monitor a kinetic of hybridization of a labeledtarget nucleotide molecule on the same capture molecule which has beenimmobilized at different defined areas of the support and which arescanned in a time dependent manner.

In a particular embodiment, the data on the quantification of theamplified target molecules performed at different PCR cycles areprocessed in order to quantify the amount of nucleotide molecule presentin the original solution before the amplification. The amplificationcycles lead to the doubling of the target sequence in each cycle whenthe efficiency of the amplification is maximal. Quantification of theoriginal nucleotide concentration is calculated from the extrapolationof the first cycle that gives a detectable value or from a valuecrossing a fixed threshold. The concentration is then calculated from areference curve or from the data obtained on a standard molecule.

In a preferred embodiment, the data are processed in order to obtain asignal value for each of the localized area. In another embodiment, thedata are processed in order to obtain a signal value for each of thelocalized area and for the local background. The data are furtherprocessed by subtracting the background from the signal value for eachof the localized area.

In a preferred embodiment, the quantification of the amount ofnucleotide molecule is performed by comparing the signal value of thelocalized area with a fixed value. In an alternative embodiment, thequantification of the amount of nucleotide molecule is performed bycomparing the number of thermal cycles necessary to reach a fixed value(cycle threshold or CT) with the CT of a reference nucleotide molecule.The reference nucleotide molecule is preferably amplified in the samesolution and detected on the same micro-array as the target nucleotidemolecule.

In another embodiment, the quantification of the amount of nucleotidemolecule is performed by comparing the number of thermal cyclesnecessary to reach a fixed value (CT) with a standard curve wherein theCTs are plotted against standard concentrations.

EXAMPLES Example 1 Preparation of a Lid Activated with Aldehyde Groups

A lid in Zeonex® having 96 defined areas sealable on a 96-wells plate(as provided in FIG. 1) was functionalized for the presence of aldehydegroups according to the following protocol.

1. Preparation of Aminated Lid

The primary amines functions were introduced into all defined areas of alid made of COC Zeonex 330R by ammonia plasma treatment. Surfacemodifications were performed by conventional low-pressure rf plasmadischarges in NH₃ plasma. The multiwell plates were positioned on trays,and the trays were placed into the chamber. The electrodes had the samesize as the trays, so that the samples were covered and treatedhomogeneously. Distance between electrode and sample was 8 cm. Afterintroduction of the samples in the reactor chamber (W 305 mm, H 300 mm,L 370 mm) and pumping down to 8×10⁻² mbar (vacuum pump: Leybold, TypeD16B), the gas flow was started and the plasma discharge was performed(working pressure 0.3 mbar, 40 kHz Generator with 30% power; dischargetime 5 min).

2. Preparation of Dextran Polyaldehyde

2.5 g of dextran (Molecular Mass 70000; Aldrich n° D1537) was dissolvedin 50 ml distilled water, then 3.594 g of potassium periodate (15.6mmol; Aldrich n′ 322423) was added. The preparation was shakenvigorously for 14 h at room temperature in the dark and dialyzed for 3days at 4° C. (cut-off of 10000; 3 times 1 litre of distilled water).The solution was centrifuged and lyophilized. 2.15 g of dextranpolyaldehyde were obtained (yield: 86%), which were stored at roomtemperature until use. 0.125 g of dextran polyaldehyde was dissolved in12.5 ml of phosphate buffer 0.1 M pH 6. The mixture was heated a fewminutes at 60° C. under vigorous stirring until complete dissolution(final solution 1%). The solution was cooled to room temperature beforeuse.

3. Preparation of Aldehyde Lid

70 μl of dextran polyaldehyde solution obtained at step 2 were added ineach defined area of the lid carrying amino groups. The lid was covedand incubated for 2 h at room temperature, then washed 3 times withdistilled water. The lid was stored under vacuum until use.

Example 2 Capture Molecule Immobilization in Defined Areas of a Lid

A lid in Zeonex® having 96 defined areas sealable on a 96-wells platewas functionalized for the presence of aldehydes according to the methoddescribed in example 1. Aminated DNA was then spotted according to amicro-array in the defined areas of the lid derivatized with aldehydegroups. The aminated capture molecules were spotted from solutions atconcentrations of 3 μM. The capture molecules were printed onto thewells using split pins (n° 1545 Genetix Limited). After the spotting,the defined areas of the lid were washed once for 1 min with 0.2% SDS,twice with distilled water. The defined areas were then incubated for 5min with NaBH₄ solution (2.5 mg/ml of PBS 75%/ Ethanol 25%), washedtwice with distilled water and dried. The lid was stored under vacuum at4° C.

Example 3 End Point Measurement of SNP in PCR Products Using a LidCarrying a Micro-array

Three PCR were performed in individual wells of a 96-well plate usingcy3 labeled primers. The first PCR was performed on a plasmid containingthe sequence of the exon 7 from CYP2C9 gene which containing themutation 3. The second PCR well was performed on a plasmid containingthe exon 5 from CYP2C9 gene which had the sequence for the wild typecorresponding to the mutation 10. The third PCR well was performed on aplasmid containing the exon 5 from CYP2C9 gene which had the sequencecontaining the mutation 10. All wells contained the primer pairs for theamplification of the sequences.

Capture molecule immobilization in a defined area of a lid

The Diaglass slides (Eppendorf, Hamburg, Germany) were functionalizedfor the presence of aldehydes according to the method described inpatent application WO02/18288. The protocol described in this patentapplication was followed for the grafting of aminated DNA to a definedarea of the aldehyde derivatized glass slide. The aminated capturemolecules were spotted from solutions at concentrations of 3 μM. Thecapture molecules were printed onto microscopic glass slides with a homemade robotic device using 250 μm diameter pins. The spots were 400 μm indiameter and the volume dispensed was about 0.5 nanolitre. Slides weredried at room temperature and stored at 4° C. until used.

The capture portion of the capture molecules used in this experiment hadthe following sequences:

The mutated base is underlined as compared to the wild type sequence.

2C9*3: (SEQ ID NO: 1, mutation 3) 5′-GGTGGGGAGAAGGTCAAGGTA-3′ 2C9*1, 3:(SEQ ID NO: 2, wild type of mutation 3) 5′-GGTGGGGAGAAGGTCAATGTA-3′2C9*1, 10: (SEQ ID NO: 3, wild type of mutation 10)5′-CTTCCTGATGAAAATGGAGAAGG-3′ 2C9*10: (SEQ ID NO: 4, mutation 10)5′-CTTCCTGATGAAAATGGGGAAGG-3′ AATSauG2 (negative hybridization control):(SEQ ID NO: 5) 5′-AACTGCTGGACTTATTTTAGGTAAGAG-3′

Each capture molecule comprised a spacer portion of 90 bases long at the5′ end of the capture portion, said spacer portion having the followingsequence:

5′ Amine-AAAGTTGAGTCCATTTGTGATGCTAGAAAAGTTGGAACTTTCTTGAACGTCTCCTATATGTCATACATGAATAGGTTGATTTTACTGTAC- 3′.

PCR and Hybridization

The CYP2C9 gene contains mutations in the exons 5 and 7. The DNAtemplate for the PCR is a plasmidic DNA obtained by cloning the entireexon 5 in vector pGEMT Easy and exon 7 in vector pCR4 Topo. They wereamplified by PCR using the following primers.

Primer pair 1, for exon 5

MP2C906: 5′-GCTTTGTACTATCAATCAGGTTGTC-3′ (SEQ ID NO: 6) MP2C902:5′-Cy3-CACAAATTCACAAGCAGTCACATAAC-3′ (SEQ ID NO: 7) Primer pair 2, forexon 7 MP2C903: 5′-Cy3-CTAAAGTCCAGGAAGAGATTGAACG-3′ (SEQ ID NO: 8)MP2C904: 5′-CAGAGTGTTGATTTGACAAGATTTTAC-3′ (SEQ ID NO: 9)

The expected sizes of the amplicons were 626 bp for CYP2C9 exon 5 and1114 bp for CYP2C9 exon 7.

The amplicon resulting from the amplification with primer pair 1 wasspecific of capture molecules SEQ ID NO: 3 or 4. The amplicon resultingfrom the amplification with primer pair 2 was specific of capturemolecules SEQ ID NO: 1 or 2.

The PCR were performed in individual tubes (200 μl) of a 96-wells plateon plasmidic DNA in a final volume of 50 μl. For the first PCR tube, thePCR mixture was the following: 1× concentrated Topo Buffer, dNTP mix(each of dNTP at a final concentration of 200 μM), 0.25 μM of primerMP2C902 Cy3 labelled at 5′ end (SEQ ID NO: 7), 0.125 μM of MP2C906 (SEQID NO: 6), 0.25 μM of primer MP2C903 Cy3 labelled at 5′ end (SEQ ID NO:8), 0.125 μM of MP2C904 (SEQ ID NO: 9), Topo Taq DNA polymerase at 2.5Uin 50 μl, potassium glutamate at 150 mM. We added 25 ng of plasmidic DNAof exon 7 of CYP2C9 carrying the mutation 3. For PCR tube 2 and 3,plasmidic DNA corresponding to exon 5 (wild type of mutation 10) and(mutation 10) of CYP2C9 were substituted to the plasmidic DNA of exon 7.The amplicon of tube 1 was supposed to react with capture molecule 2C9*3(SEQ ID NO: 1), amplicon of tube 2 with capture molecule 2C9* 1,10 (SEQID NO: 3) and amplicon of tube 3 with capture molecule 2C9*10 (SEQ IDNO: 4).

A glass lid comprising a defined area having fixed upon its surface amicroarray was fixed on the top of the three PCR tubes by means of anadaptor. The PCR were performed in a thermocycler (Eppendorf, Hamburg,Germany). Samples were first denatured at 94° C. for 5 min. Then 40cycles of amplification were performed consisting of 30 sec at 94° C., 1min at 63° C. and 1 min at 72° C. and a final extension step of 10 minat 72° C.

After the PCR, the multiwell plate was flipped and the content of threewells comprising three different PCR products (50 μl each) was contactedwith the lid carrying immobilized capture molecules and incubated for 45min at 60° C. After hybridization, the multiwell plate was flipped back,centrifuged for 1 min at 1440 rpm to remove the PCR solution from thelid and the micro-array was read in fluorescence using the Axon scanner(4100 personal). Scanning was performed with the 532 channel for Cy3detection at a gain of 600 with a resolution of 20 micrometer.

The scanner used as excitation light a laser which was focussed on thesurface of the support. The emission light was detected and amplified bya photomultiplier. After image acquisition, the scanned 16-bit imageswere imported to the software, “Genepix 5” (Axon, Union city, Calif.,USA) which was used to quantify the signal intensities. The signal wasquantified on four capture molecules present in three replicates on thearray: 2C9* 1,3 (SEQ ID NO: 2), 2C9*3 (SEQ ID NO: 1), 2C9*1,10 (SEQ IDNO: 3) and 2C9*10 (SEQ ID NO: 4). The local background was subtractedand signal minus background was plotted against the capture molecules.The arrays also contained a negative hybridization control (neg hyb ctl,SEQ ID NO: 5), and positive detection control labeled with Cy3 presentin quadruplicate on the array. The capture molecule used as negativehybridization control was silent.

Result of the end point measurement of SNP in PCR products on the lid ispresented in FIG. 4. The result shows signals on the specific capturemolecules SEQ ID NO: 1, 3 and 4 as expected. There was a very smallsignal on the wild type capture molecule SNP2C9* 1,3 (SEQ ID NO: 2). Thesignal value was 80 compared to 7303 for the mutated capture molecule2C9*3 (SEQ ID NO: 1). This value represents only 1% of the positivevalue. This is due to a small cross reaction of the amplicons with thewild type sequence (SEQ ID NO: 2) due to the close homology of sequenceof the capture molecules SEQ ID NO: 1 and 2. The negative control ofhybridization (neg hyb ctl) was negative.

For the signal on the mutated capture molecule SNP2C9*10, the value was11208 and 6938 for the corresponding wild type capture moleculeSNP2C9*1,10.

Thus, the invention has been described by reference to certainembodiments discussed above. It will be recognized that theseembodiments are susceptible to various modifications and alternativeforms well known to those of skill in the art.

Many modifications in addition to those described above may be made tothe structures and techniques described herein without departing fromthe spirit and scope of the invention. Accordingly, although specificembodiments have been described, these are examples only and are notlimiting upon the scope of the invention.

1. A sealable lid for a PCR multiwell plate provided with at least one defined area, said defined area being configured to be mated to at least one well of the multiwell plate, at least one of said defined area being pretreated to be provided with unlabeled capture molecule.
 2. The lid of claim 1, wherein at least one defined area bears an immobilized unlabeled capture molecule.
 3. The lid of claim 2, wherein the capture molecule is a polynucleotide.
 4. The lid of claim 2, wherein different defined areas of the same lid have fixed the same capture molecules. 5 The lid of claim 1, wherein the lid contains at least 2, preferably at least 6, more preferably at least 24, still more preferably at least 96 defined areas.
 6. The lid of claim 1, wherein the multiwell plate has at least 2, preferably at least 8, more preferably at least 24, still more preferably at least 96, even more preferably at least 384 wells.
 7. The lid of claim 1, wherein the wells of the multiwell plate which are separated from each other by a pitch of 4.5 mm or a multiple thereof.
 8. The lid of claim 1, wherein the measurement of the amplified target nucleotide molecules is performed on the lid of the multiwell plate after hybridization on capture molecules.
 9. The lid of claim 1, wherein the lid is used for real-time PCR measurement.
 10. The lid of claim 1, wherein the lid is used for end point PCR measurement.
 11. The lid of claim 2, wherein the defined area has fixed upon its surface at least 4 different capture molecules being physically separated in compartments.
 12. The lid of claim 2, wherein the defined area has fixed upon its surface a micro-array comprising at least 5 different capture molecules being immobilized in specifically localized areas of said defined area.
 13. The lid of claim 12, wherein the micro-array comprises more than 10 different capture molecules, preferably more than 20 capture molecules, more preferably more than 50 capture molecules.
 14. The lid of claim 1, wherein the defined area has aldehyde groups fixed upon their surface.
 15. The lid of claim 14, wherein the defined area is divided in at least 4 compartments.
 16. The lid of claim 15, wherein the defined area is used to fix capture molecules terminated by a primary amino group.
 17. The lid of claim 1, wherein the lid is maintained flat at temperature higher than 85° C.
 18. The lid of claim 1, wherein the defined area has a flatness tolerance of less than 100 microns and better of less than 25 microns.
 19. The lid of claim 1, wherein the light transmittance of the lid at the wavelength used for the detection is higher than 80%, preferably higher than 90%.
 20. The lid of claim 1, wherein the lid comprises or is made of a material selected from the group consisting of glass, metal and polymer.
 21. The lid of claim 20, wherein the polymer is selected from the group consisting of: polycarbonate (PC), polyethylene (PE), Cycloolefin copolymer (COC), cyclic olefin polymer (COP) or a mixture thereof.
 22. The lid of claim 1, wherein the defined area is configured to be mated to at least one well of the multiwell plate by means of a sealable material.
 23. The lid of claim 1, wherein the defined area is configured to be mated to at least one well of the multiwell plate by means of an adaptor comprising a sealable material.
 24. The lid of claim 22, wherein the sealable material is made of a soft polymer or a glue.
 25. A sealable lid (1) for a PCR multiwell plate provided with at least one defined area, said defined area being configured to be mated to at least four wells of the multiwell plate, at least one of said defined area being pretreated to be provided with unlabeled capture molecule.
 26. The lid of claim 25, wherein the lid contains at least 2, preferably at least 6, more preferably at least 24, still more preferably at least 96 defined areas.
 27. The lid of claim 25, wherein the multiwell plate has at least 2, preferably at least 8, more preferably at least 24, still more preferably at least 96, even more preferably at least 384 wells.
 28. The lid of claim 25, wherein the wells of the multiwell plate which are separated from each other by a pitch of 4.5 or 9 mm.
 29. The lid of claim 25, wherein the lid is used for end point PCR measurement.
 30. The lid of claim 25, wherein the defined area has aldehyde groups fixed upon their surface.
 31. The lid of claim 25, wherein the defined area is divided in at least 4 compartments.
 32. The lid of claim 25, wherein the defined area is used to fix capture molecules terminated by a primary amino group.
 33. The lid of claim 25, wherein the lid is maintained flat at temperature higher than 85° C.
 34. The lid of claim 25, wherein the defined area has a flatness tolerance less than 100 microns and better less than 25 microns.
 35. The lid of claim 25, wherein the light transmittance of the lid at the wavelength used for the detection is higher than 80% preferably higher than 90%.
 36. The lid of claim 25, wherein the lid comprises or is made of a material selected from the group consisting of glass, metal, polymer.
 37. The lid of claim 36, wherein the polymer is selected from the group consisting of: polycarbonate (PC), polyethylene (PE), Cycloolefin copolymer (COC), cyclic olefin polymer (COP) or a mixture thereof.
 38. The lid of claim 25, wherein the defined area is configured to be mated to at least one well of the multiwell plate by means of a sealable material.
 39. The lid of claim 25, wherein the defined area is configured to be mated to at least one well of the multiwell plate by means of an adaptor comprising a sealable material.
 40. The lid of claim 38, wherein the sealable material is made of a soft polymer or a glue. 